We have continued our study of the structure of chromatin, the complex of proteins and DNA found in eukaryotic nuclei. Our earlier work suggested that the DNA of the nucleosome is not held in place on the surface of the particle by interactions between histones and bases. We have now shown that a considerable part of the DNA is held in place by only a few electrostatic interactions between histones and phosphodiester groups. We have also examined the 30 nm fiber formed when nucleosomes pack together in the nucleus, and determined the orientation of the nucleosomes in the fiber. We have studied the binding of histones and HMG proteins to nucleosomes, and discovered properties that may be related to higher order structure formation and to gene activation. We have developed a cell-free system for studying RNA polymerase II-dependent transcription of DNA and chromatin.